1. Field of the Invention
The present invention relates to a D-amino acid aminotransferase mutant, a nucleic acid sequence encoding such enzyme mutant, an expression vector containing such nucleic acid sequence, the preparation of such enzyme mutant and the use thereof. The invention also provides a method for selecting clones with high D-amino acid aminotransferase activity.
2. Description of the Related Prior Art
D-amino acid aminotransferase (DAT) is widely used in the transamination reaction and the production of D-amino acid. DAT has high stereoselectivity and can convert one amino acid to another in the presence of a pyridoxal or pyridoxamine phosphate and an alpha-keto acid corresponding to the amino acid to be produced.
DATs from various sources have been isolated and characterized. DAT was firstly found in the extracts of Bacillus subtillis by Thorne et al., (1954), J. Bacteriol. 69: 357-362. Subsequently, DAT was demonstrated in bacteria of the genera Bacillus (Tanizawa, K. et al., (1989), J. Biol. Chem. 264, 2445-2449; Thorne, C. B. and D. M. Molnar, (1955), J. Bacteriol. 70, 420-426), Rhodospirillum rubrum (Hug, D. H. et al., (1957) Arch. Biochem.Biophys. 72, 369-375), Rhizobium japonicum (Gosling, J. P. et al., (1978) Biochim. Biophys. Acta 522, 84-95), and Staphylococcus haemolyticus (Pucci, M. J. et al., (1995) J. Bacterol. 177, 336-342), higher plants and mammalian livers.
A D-amino acid aminotransferase has been isolated from Bacillus sphaericus. The enzyme has a molecular weight about 60 kDa and consists of two identical subunits. The encoding gene was isolated, sequenced and cloned into the plasmid pIF1001 and transformed into E. coli to express the enzyme (Ian G. F. et al., (1998), Journal of Bacteriology, P. 4319-4323). The optimum pH for the reaction of the Bacillus sphaericus D-amino acid aminotransferase is 8.5.
D-amino acid aminotransferase is useful for producing various D-amino acids which can be used in industrial or pharmaceutical products such as sweeteners, antibiotics, peptide enzymes and peptide hormones. For example, Alitame, tyrocidin A, antinomycin D, penicillin N and cephalosporin C, and cyclosporin A comprise D-alanine and D-phenylalanine, D-valine, D-alpha amino valerate, D-alpha aminoavalerate and D-alanine, respectively. Short peptides can also be capped with D-form amino acid to prevent digestion by protease and slow down enzymatic degradation. It is disclosed in U.S. Pat. No. 4,745,061 that Bacillus lichiformis has two aminotransferase: D-aminoacid transaminase and cephalosporin C aminotransferase. As shown in column 1 of the patent, the D-aminoacid transaminase is not capable of converting cephalosporin C. The cephalosporin C aminotransferase can be used in the conversion of cephalosporin C (Ceph C) to Glutaryl-7-aminocephalosporanic acid (GL-7ACA) which is a highly valuable pharmaceutical chemical for the synthesis of cephem antibiotics.
One objective of the invention is to provide a mutant D-amino acid aminotransferase which can simultaneously produce Glutaryl-7-aminocephalosporanic acid and D-amino acid.
Another objective of the invention is to provide a nucleic acid compound encoding the mutant D-amino acid aminotransferase of the invention, an expression vector comprising said nucleic acid and a host cell transformed with said vector.
The invention also provides a method for selecting clones with high D-amino acid aminotransferase activity.